1. Field of the Invention
The invention is a reagent, methods and kits for the isolation of RNA from RNA containing cells and tissues, and preferably from cells and/or tissues from plants and plant materials.
2. Related Art
Commercial reagents and kits available for the isolation of RNA do not accommodate difficult specimens, especially those specimens rich in polyphenolics (e.g., conifer needles) or starch (e.g., potato tuber or seeds). RNA yields from these reagents and kits are low, or RNA quality is poor as demonstrated by low A260/280 ratios or gel electrophoresis.
Several methods are described in the literature for the isolation of RNA from pine needles and spruce needles that are reported to give RNA of good quality. See, e.g., Schneiderbauer, A. et al, Isolation of Functional RNA from Plants Rich in Phenolic Compounds Analytical Biochemistry 197:91-95 (1991); Graham, Glenn C., A method of extraction of total RNA from Pinus radiata and other conifers, Plant Molecular Biology Reporter, 11:32-37 (1993); Chang, Shujun, Puryear, Jeff and Cairney, John, A simple and efficient method for isolating RNA from pine trees, Plant Molecular Biology Reporter 11:113-116 (1993); and Bahloul, Mouna and Burkard, Gerard, An improved method for the isolation of total RNA from spruce tissues, Plant Molecular Biology Reporter 11:212-215 (1993).
However, all of these known methods are extremely laborious. For example, Schneiderbauer extracts pine or spruce specimen with acetone at −70° C. to remove polyphenolics. The pellet is then homogenized in the presence of 0.1% (v/v) TRITON®X-100 (octyl phenol polyethoxylate), 15 mM DTT (dithiothreitol) and phenol. The homogenization process releases RNA, DNA, and proteins. Proteins are removed by phase separation in an organic extraction phase. Then, DNA is removed by centrifugation on a cesium chloride cushion.
Another method (Graham) uses guanidinium isothiocyanate to disrupt the tissue, and RNA is then recovered by centrifugation on a cesium trifluoroacetate cushion. Other methods use cationic (Chang, et al) or anionic (Bahloul, et al) detergents to release the nucleic acids followed by either multiple alcohol precipitation or phenol extraction, and lithium chloride precipitation to remove DNA from the isolated RNA.
The reagent and methods of the present invention simplify the RNA extraction process, yielding high quality RNA from RNA containing materials, especially ordinary plant specimens and those enriched in polyphenolics and starch.